1. Field of the Invention
The present invention relates to imaging that accomplishes extended depth of field and 3D ranging simultaneously. In particular, the present invention accomplishes such ranging utilizing an engineered point spread function and forming dual images for processing.
2. Discussion of Related Art
Breakthroughs in optoelectronic technologies over the past few decades have led to an explosion of new microscope configurations for addressing specific biological problems. However, these microscopes still have significant shortcomings that, if overcome, could provide biologists with the ability to conduct a greater range of live-cell imaging investigations. Specifically, the ability to create images of sharply focused fine structure throughout the entire 3D cell volume at video rates or faster is still needed. Also needed is a way to reduce the ubiquitous noise common to low light imaging applications, such as in live-cell fluorescence studies, even when using the best available cameras and sensors. A final need is to make microscope systems that are less complicated, less costly and therefore accessible to many more biologists, without compromising the high performance capabilities provided by these modern high-end instruments. Our proposed new microscope will therefore aim to provide significant advancements toward addressing these needs.
Limitations of existing microscopes: For over 400 years, the compound optical microscope has been a primary tool for imaging the world of living systems. Although improvements in the past few decades have generated many new capabilities (e.g. confocal, multi-photon, widefield deconvolution, specific fluorescence techniques such as FLIM, STORM, PALM, to name a few) the underlying optical principles governing the design of objective lenses used by all of these microscope systems have remained unchanged. These design principles are directed toward producing the best possible lateral resolution in each recorded image through maximizing objective numerical aperture (NA) and correcting all common optical aberrations. Strict adherence to these design criteria results in well-known tradeoffs or compromises in objective lens performance, specifically: (1) increasing lateral resolution (i.e. increasing NA) causes a rapid decrease in image depth of field so that many features inside thick objects may appear blurred if they fall above or below the plane of best focus, and (2) each single image is a two-dimensional (2D) projection in which the 3D positions of biological features inside the specimen volume are lost.
Confocal and widefield deconvolution microscopes overcome these two tradeoffs by moving the plane of best focus up and down throughout the specimen volume to acquire a stack of “best focused” images for further processing. However, this process is slow, usually requiring a minimum of 1-2 seconds to acquire each image stack which can lead to increased fluorophore bleaching as well as motion blur of fast moving cell features. Other schemes, such as multi-focal plane illumination designs that try to speed up the depth scanning process, introduce the possibility of alignment errors and require expensive optoelectronics.
A technique called wavefront coding has been used in an attempt to increase depth of field of images in optical microscope systems. A waveplate (for example as taught in U.S. Pat. No. 5,748,371) in the optical path creates image point spread functions with increased depth of focus. However, the wavefront coding approach was so severely limited by noise and image reconstruction artifacts that it proved to be an unviable option for high-resolution, extended depth of field (EDF) biological microscopy.
A further limitation of these microscopes is that the only way they address the problem of reducing image noise is by using very expensive, low-noise detectors such as EMCCD or CMOS cameras, or photomultiplier tubes in the case of confocal point scanning systems. A need remains for the reduction or elimination of image noise artifacts. These usually appear as randomly scattered bright image pixels arising from detector electronics and shot noise, and are particularly problematic in light starved applications such as when observing weakly fluorescing live-cell structures. In these situations, detector noise of various types can be so prevalent as to mask the tiny features of biological interest when imaging near the resolution limit of the microscope.
A need remains in the art for an imaging system that simultaneously increases depth of field and encodes 3D ranging information, while reducing image noise.